Protein kinases are critical in regulating the activity of ion transport mechanisms involved in salt and fluid reabsorption in the kidney, control of blood pressure, and modulation of inhibitory synaptic transmission in the peripheral nervous system. SPAK, OSR1, WNK kinases, and possibly some PKC isotypes are all involved in modulating cation-chloride cotransporter function. SPAK and OSR1, in particular, directly activate a Na-K-2Cl cotransport (NKCC) mechanism involved in accumulating Cl- in primary afferent neurons. This Cl- accumulation facilitates primary afferent depolarization, presynaptic inhibition, and the filtering of sensory noise. The precise mechanisms by which these kinases act on NKCC1 function are not fully understood. To better delineate the molecular mechanisms and the functional significance of kinase regulation of NKCC1 function, we propose to examine the role of SPAK/OSR1 in vivo through the use of various mouse models. We will determine whether tissue-specific deletionofOSR1 affects NKCC1 function in primary afferent neurons. We will determine whether different signals that lead to NKCC1 phosphorylation involve the activation of SPAK and/or OSR1. We also propose to examine the identity of the kinases (e.g. WNKs and PKCs) that act upstream of SPAK and OSR1 in a rat primary afferent neuronal cell line. This will be accomplished by examining expression of the upstream kinases in dorsal root ganglion neurons, assessing their roles in heterologous expression, and eliminating their expression through shRNA silencing techniques. Finally, we will examine the molecular details of the interaction between SPAK/OSR1 and NKCC1, analyze and characterize the role of a putative autoinhibitory domain, and determine the role of swapped domain dimerization in the activation of SPAK/OSR1. This will be achieved by measuring protein-protein interaction in vivo and in vitro, and functionally examining the consequence of mutagenesis of specific regions of the proteins. These studies will provide valuable new insights regarding the mechanism of NKCC1 activation by kinases, and lead to a better understanding of the role of cation-chloride co transporter activity in the gating of sensory information from the peripheral to the central nervous system. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because these studies will provide mechanistic insights into the regulation of ion transport mechanisms involved in renal function, hypertension, and neuropathic pain. Our application is focused at understanding the critical aspects of primary afferent depolarization and the gating of chronic pain signals. Our proposal is relevant to the part of NIH's mission to pursue fundamental knowledge which will extend healthy life and reduce the burdens of illness and disability.